A jumbo phage that forms a nucleus-like structure evades CRISPR–Cas DNA targeting but is vulnerable to type III RNA-based immunity

Abstract

CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages 1 . However, DNA modification 2,3 , the production of anti-CRISPR proteins 4,5 and potentially other strategies enable phages to evade CRISPR-Cas. Here we discovered a Serratia jumbophage that evaded type I CRISPR-Cas systems, but was sensitive to type III immunity. Jumbophage infection resulted in a nucleus-like structure enclosed by a proteinaceous phage shella phenomenon only reported recently for distantly related Pseudomonas phages 6,7 . All three native CRISPR-Cas complexes in Serratia -type I-E, I-F and III-A -were spatially excluded from the phage nucleus and phage DNA was not targeted. However, the type III-A system still arrested jumbophage infection by targeting phage RNA in the cytoplasm in a process requiring Cas7, Cas10 and an accessory nuclease. Type III, but not type I, systems frequently targeted nucleus-forming jumbophages that were identified in global viral sequence datasets. These findings explain why many bacteria harbour both RNA-and DNA-targeting CRISPR-Cas systems 1,8 . Together, our results indicate that jumbophage nucleus-like compartments serve as a barrier to DNA-targeting, but not RNA-targeting defences, and that this phenomenon is widespread amongst jumbophages.

Multisubunit RNA Polymerases of Jumbo Bacteriophages

“…The genome of jumbo phage AR9 infecting B. subtilis , a close relative of the PBS2 phage, was sequenced and shown to contain genes orthologous to the RNAP genes of phiKZ-like phages [ 18 ], suggesting that the PBS2 RNAP purified a long time ago [ 35 , 36 ] in fact belongs to this new group of non-canonical jumbo phage RNAPs. At the time of this writing, several dozens of jumbo phages encoding distant homologs of the β′ and β subunits are known [ 18 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. They infect diverse bacterial hosts including members of Pseudomonas , Salmonella , Yersinia , Erwinia , Vibrio , Ralstonia , Bacillus , Aeromonas , Serratia , Klebsiella , Escherichia , and other genera [ 18 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , …”

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…3 (15)). Furthermore, it has been proposed that jumbo phages encapsulate their genome in a nucleus-like proteinaceous shell upon cell entry to evade CRISPR-Cas 128 . Similar strategies including synthetic physical encapsulation or co-localization of enzymes via scaffold molecules have already proven effective to protect molecules from degradation by host enzymes and, moreover, to increase metabolic flux and reduce cross-talk 122 , 123 .…”

How important is CRISPR-Cas for protecting natural populations of bacteria against infections by mobile genetic elements?

“…Some "jumbophages", which are a class of phages with genome sizes that exceed 200kb, have been reported to form nucleus-like structure during infection (120)(121)(122). These structures contain the phage genomes, which shields it from DNA-targeting CRISPR-Cas systems, but not from systems that have RNAtargeting activity, such as Type III-A and VI-A CRISPR-Cas (123,124). This variation in the level of protection explains why in nature spacer acquisition from nucleus-forming jumbophages is detected more frequently for bacteria that carry Type III systems compared to those that carry Type I-E or I-F systems (123).…”

“…These structures contain the phage genomes, which shields it from DNA-targeting CRISPR-Cas systems, but not from systems that have RNAtargeting activity, such as Type III-A and VI-A CRISPR-Cas (123,124). This variation in the level of protection explains why in nature spacer acquisition from nucleus-forming jumbophages is detected more frequently for bacteria that carry Type III systems compared to those that carry Type I-E or I-F systems (123). A lack of protection by CRISPR immunity is not limited to jumbophages: E. coli strains carrying Type I-E CRISPR-Cas that were engineered to carry a single targeting spacer against different phages revealed a lack of protection against phages R1-37 (a giant phage) and T4 (125).…”